The objective of this proposal is to characterize the development of antigen-specific memory Th cells in an experimental mouse model of Th cell immunity. Antigen specific Th cells have been difficult to study in normal animals due to the low frequency of Ag specific cells. Conventional analysis of populations of cells are limited by the presence of contaminating cells. Purification of Ag specific cell populations may still be heterogenous with respect to function. This proposal seeks to avoid these confounding aspects by direct analysis of purified Ag specific cells at the single cell level. This will allow for direct analysis of TCR specificity, as well as surface molecule expression and expression and secretion of cytokines. Comparison of Ag specific primary effector cells, resting memory cells and memory effector cells will clarify the cellular and molecular dynamics of these populations.